RNA Decoys for DNA Binding Proteins

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There is growing appreciation that small, non-coding RNAs can participate in gene regulation. Can small RNAs inhibit DNA-binding proteins? We have developed an artificial example by performing in vitro genetic selection experiments identifying a small RNA aptamer that competitively inhibits human transcription factor NF-kappaB binding to DNA in vitro. Optimization by yeast in vivo genetic selections resulted in an RNA that inhibits NF-kappaB in living yeast cells. We have solved the X-ray co-crystal structure of this unusual RNA/NF-I<B complex. This structure demonstrates how RNA structural plasticity allows DNA mimicry, suggests a new strategy for engineering RNAs specific for different NF-kappaB dimers, and identifies a novel "open" NF-I<B conformation incompatible with DNA binding. NF-kappaB plays an essential role in regulating genes involved in inflammatory responses and protects tumor cells from apoptosis, thus limiting the effectiveness of chemotherapy and radiotherapy. Agents that reduce NF-kappaB activity tend to promote apoptosis and have shown promising anticancer activities. Using NF-kappaB as a model of therapeutic interest, we will optimize and test anti-NF-kappaB RNA aptamers in bacterial, yeast, and human cell culture experiments. We will also determine the structure of the free RNA aptamer to understand conformational changes that occur upon protein binding. Our unprecedented RNA/NF-kappaB crystal structure now allows virtual drug screens and in vitro and in vivo validation assays for novel NF-KappaB inhibitors. Such drugs would act by stabilizing our newly-discovered "open" NF-kappaB conformation that is incompatible with DNA binding. Four specific aims are proposed: Aim 1. Optimize and extend anti-NF-kappaB RNA aptamer function by genetic selections in bacteria and yeast. Aim 2. Analyze the structural basis for NF-kappaB binding by anti-NF-kappaB RNA aptamers. Aim 3. Analyze NF-kappaB inhibition by anti-NF-kappaB RNA aptamers in cultured mammalian cells. Aim 4. Identify small molecules that inhibit DNA binding by NF-kappaB.